Myosin Va, a class V processive motor, transports tubules of smooth endoplasmic reticulum (SER) into the dendritic spines of cerebellar Purkinje neurons (PNs). These SER tubules provide the source of calcium downstream of mGluR1 activation that drives synaptic plasticity at parallel fiber: PN synapses. Calcium release from SER tubules is mediated by the IP3 receptor (type 1 inositol 1,4,5-trisphosphate receptor or IP3R1), a resident SER protein. The focus of this study was to identify the receptor on the surface of the SER for myosin Va. A yeast two-hybrid screen and immunoprecipitation analyses identified two potential receptor components that interact with both myosin Va and IP3R1: phospholipase C beta 4 (PLC&#946;4) and receptor-for-activated-kinase 1 (RACK1). Initial work has shown that both PLC&#946;4 and RACK1 are enriched in dendritic spines, and live cell imaging has shown that RACK1, like myosin Va, localizes at the tip of SER tubules that are moving into spines. Furthermore, the dendritic spines of PNs from IP3R1-null mice are devoid of SER, arguing that IP3R1 is required for the myosin Va-dependent translocation of SER into spines. Finally, the enrichment of PLC&#946;4 and RACK1 in dendritic spines is disrupted in both myosin Va knockdown cells and IP3R1-null cells. Together, these data suggest that myosin Va may be recruited to the SER via the IP3 receptor, with PLC&#946;4 and/or RACK1 providing a bridge linking the myosin to the receptor. Myosin Va, a processive class V motor, is involved in the transport of diverse cargos, including melanosomes, endoplasmic reticulum (ER), and mRNAs. The creation of tools to identify proteins that interact with myosin Va should increase our understanding of the cellular processes supported by this myosin. Towards that end, we report here the generation of a tandem affinity purification (TAP) tag knockin mouse at the MYO5A locus. A recombineering-based approach was used to insert via homologous recombination a TAP-tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and the FLAG epitope tag into MYO5A locus immediately after the initiation codon. Mice homozygous for the knockin allele, which express the TAP-tagged version of myosin Va (TAP-MyoVa) exclusively and under the control of the endogenous MYO5A promoter, exhibit normal coat color and no evidence of ataxia, arguing that TAP-MyoVa functions normally. Consistently, the dendritic spines of Purkinje neurons isolated from this mouse are fully loaded with ER, in contrast to the spines of Purkinje neurons from dilute (myosin Va null) mice, which are devoid of ER. Similarly, melanosomes are distributed normally in melanocytes from the TAP-tagged myosin Va mouse, in contrast to melanocytes from dilute mice, where the organelles are concentrated in the cell center. Moreover, introduction of a CMV promoter-driven TAP-Tag myosin Va construct into dilute melanocytes rescues melanosome distribution. Given this clear evidence that TAP-MyoVa is fully functional, we purified TAP-MyoVa and associated proteins directly from juvenile mouse cerebella excised from TAP-tagged mice and subjected the samples to mass spectroscopic analyses. Importantly, elutes contained several known myosin Va binding partners (list), further verifying that TAP-MyoVa is fully functional. Moreover, we found numerous novel interacting proteins. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this myosin in vivo. Dendritic spines are small, actin-rich protrusions on the surface of neuronal dendrites that serve as sites of excitatory synaptic input and memory formation. Previous studies have implicated myosin II, the conventional non-muscle myosin, in spine organization and function. Here we have explored the localization within cerebellar Purkinje neurons (PNs) of myosin 18A (M18A)-alpha and M18A-beta, two spliced isoforms of M18A, a recently discovered member of the myosin super family that is similar to, and yet distinct from, class II myosins. These distinctions include a C-terminal PDZ-ligand motif in both spliced isoforms, and in the case of M18A-alpha, a 300-residue N-terminal extension that harbors an apparent ATP-independent actin-binding site, a Lys- and Glu-rich region (KE region), and a PDZ domain. The fact that M18A-beta lacks this N-terminal extension suggests distinct functions for these two isoforms. Using GFP-tagged versions of M18A-alpha and beta and a novel system for gene transduction, we find that M18A-alpha, but not M18A-beta, localizes dramatically to the dendritic spines of PNs in dissociated culture. Moreover, M18A-alphas spine distribution overlaps extensively with that of F-actin, as revealed using a novel reporter for F-actin (F-Tractin). The postsynaptic, spine localization of M18A-alpha was confirmed by immuno-EM of cerebellar tissue sections using a highly specific anti-M18A antibody. Importantly, M18A-alphas N-terminal extension by itself targets extensively to spines, arguing that it is largely if not entirely responsible for the myosins spine localization. Function-blocking point mutations show that the KE region and the PDZ domain within M18A-alphas N-terminal extension do not determine spine targeting. Rather, the interaction of the ATP-independent actin-binding site with actin filaments appears to drive the spine localization of M18A-alpha. Consistently, in vitro binding assays using M18A-alphas N-terminal extension show that it binds F-actin with moderately high affinity (305 105 nM). We suggest that M18A-alpha may play important roles in the development, organization, and/or function of PN spines. Future experiments using a conditional M18A knockout mouse, which we have successfully created, in combination with the PN-specific expression of Cre recombinase, will alow us to address the physiological significance of the myosin's spine localization in PNs.